DESCRIPTION: Among all epithelial tissues, the corneal epithelium is most unique in the easy access of its stem cells (SCs) in the limbal region separable from its transient amplifying cells (TACs). In the last funding period, we have gathered strong laboratory and preclinical data supporting the hypotheses that ex vivo expansion of limbal epithelial SCs can be achieved by culturing on amniotic membrane (AM), and that such an ex vivo expanded tissue can be used for a short term to maintain a limbal epithelial phenotype after engraftment to nude mice, and for a long term to restore a normal corneal surface after engraftment to rabbit limbal deficient corneas. To exploit further potentials of limbal SCs, it is important to understand how SC fate decision in self renewal and progeny production is regulated by its niche. This application explores the regulatory mechanism of TGF-beta signaling by TACs in a serum-free, fibroblast-free and matrix-free niche (Aim 1), by amniotic epithelial cells and stroma in AM as a surrogate (ex vivo) niche (Aim 2), and by limbal fibroblasts in the limbal stroma as the native (in situ) niche (Aims 3 and 4). Completion of these studies will shed new light on how a progeny may exert a feedback control on SC fate. The novel method of isolating and expanding limbal SCs by eliminating TAC's influence will help isolate other SCs in the body, identify potential SC marker(s), and can also be used as a new cell source for tissue engineering in the future. Furthermore, amniotic epithelial cells may be used as a feeder layer and HA-containing stroma matrix may be used to substitute AM to expand limbal SCs in the future. Limbal fibroblasts can be impregnated on the stromal side of AM as a new strategy of bio-engineering a surgical graft to replace the limbal tissue. The role of "irreversible epithelial-mesenchymal transition" of limbal SCs into fibroblasts and myofibroblasts radically changes our view of how limbal stem cell deficiency develops under inflammation. The new knowledge thus gathered will lead us to develop other new surgical reconstructive strategies of treating corneal blinding diseases inflicted by limbal stem cell deficiency such as chemical/thermal burns, Stevens Johnson syndrome (Toxic Epidermal Necrolysis) and a variety of cicatricial surface disorders, as well as novel therapeutics in regenerative medicine and tissue engineering based on adult SCs and their novel matrix niches in the future.